Modified fatty acids (mFAs) (also referred to as unusual or specialized fatty acids) obtained from plants have important roles as feedstock for industrial materials such as lubricants, protective coatings, plastics, inks, cosmetics, and etc. mFAs are naturally produced by a limited number of species (source plants) which are generally not readily cultivated at scale. The potential for industrial use of mFAs has led to considerable interest in exploring their production in transgenic crop plants rather than isolating, for example, a specific mFA from a specific source plant. Unfortunately, transgenic crop plants transformed with source plant genes or DNA sequences (e.g., cDNAs) encoding mFA-producing enzymes generally accumulate only modest amounts of the mFA compared to amounts accumulating in the natural source plant (Napier (2007) Ann. Rev. Plant Biol. 58:295-319). Levels of mFAs accumulating in the seeds of transformed plants rarely exceed 20% of the total seed FA whereas, for example, castor seeds naturally accumulate >90% ricinoleic acid and Tung (Aleuites fordii) seeds naturally accumulate >80% α-Eleostearic acid (Drexler et al. (2003) J. Plant Physiol. 160:779-802; Thelen et al. (2002) Metab. Eng. 4:12-21).
In order to elevate the content of mFAs in the engineered transgenic plants to levels approaching that found in the source plant, it is necessary to 1) optimize the synthesis of mFA (Mekhedov, et al. (2001) Plant Mol. Biol. 47:507-518) 2) minimize its degradation (Eccleston et al. (1998) Plant Cell 10:613-621); and 3) optimize its incorporation into triacylglycerol (TAG) (Bafor et al. (1990) Biochem. J. 272:31-38; Bates et al. (2011) Plant J. 68:387-399); van Erp et al. (2011) Plant Physiol. 155:683-693).
Among the modified fatty acids, cyclic FAs (CFAs) (generally cyclopropane- and cyclopropene-containing FAs (CPAs)) are desirable for numerous industrial applications. The strained bond angles of the carbocyclic ring contribute to their unique chemical and physical properties. Hydrogenation of a cyclic FA results in ring opening to produce a methyl-branched FA. Branched-chain FAs are ideally suited for the oleochemical industry as feedstocks for the production of lubricants, plastics, paints, dyes, and coatings (Carlsson et al. (2011) Eur. J. Lipid Sci. Technol. 113:812-831).
Cyclic FAs (CFAs) have been found in certain gymnosperms, Malvales (including cotton), Litchi and other Sapindales. They accumulate to as much as 40% in seeds of Litchi chinensis (Gaydou et al. (1993) J. Agri. & Food Chem. 41:886-890; Vickery (1980) J. Amer. Oil Chem. Soc. 57:87-91). Sterculia foetida accumulates a desaturated cyclic FA, cyclopropene FA (sterculic acid), to >60% of its seed oil.
In all cases examined, the production of a CPA begins with methyl group addition by a cyclopropane fatty acid synthase (CPS) enzyme at a carbon-carbon double bond of an unsaturated fatty acid compound. For example, the first step in the synthesis of sterculic acid is the formation of the CPA, dihydrosterculic acid (DHSA), by the CPS enzyme that transfers a methyl group from S-adenosylmethionine to C9 of the oleoyl-phospholipid followed by cyclization to form the cyclopropane ring and dehydrogenation to form the cyclopropene fatty acid, sterculic acid (Bao et al. (2002) Proc. Natl. Acad. Sci. USA 99:7172-7177; Bao et al. (2003) J. Biol. Chem. 278:12846-12853; Grogan et al. (1997) Microbiol. Mol. Biol. Rev. 61:429-441).
Because none of the known natural source plants for CPAs are suitable for commercial-scale cultivation it is desirable to create a crop plant and preferably an oilseed crop plant that accumulates high levels of CPA by expressing a heterologous CPS in the crop plant seeds. However, to date, heterologous expression of plant cyclopropane synthase coding sequences led to only 1 to 3% DHSA in transformed tobacco (K. M. Schmid, U.S. Pat. No. 5,936,139) and only −1.0% CPA in transgenic seeds (Yu et al. (2011) BMC Plant Biol. 11:97). Thus merely expressing a cyclopropane synthase coding sequence in a crop plant is insufficient to generate a transformed crop plant to produce industrially meaningful amounts of CPAs.
As noted, and as exemplified in the results for CPA, the engineering of transgenic crop plants that accumulate commercially meaningful amounts of a modified fatty acid compound is a complex proposition requiring a refined balance of synthesis, degradation and conversion to triacylglycerol storage compounds. Mere over-expression of the “modified fatty acid synthase” or “fatty acid modifying” coding sequence has proven insufficient.
Significant efforts to achieve this balance have been devoted to generating transgenic crop plants that accumulate commercially relevant amounts of ricinoleic acid. Ricinoleic acid production has been targeted because of its well-known industrial utility and the difficulties associated with obtaining it from the seeds of castor. The combination of Smith, et al. (2003) Planta 217:507-516, van Erp, et al. (2011), and Browse, et al. (U.S. Pat. No. 8,101,818), the entire contents of all three of which are incorporated herein by reference, serves to frame the issues.
One such issue is in part described in Smith, et al. (2003) where the influence of the genetic or phenotypic background of the progenitor parent plant is considered. The authors of that work explore the effects of several parental backgrounds, including plants deficient in FAD2 activity, FAE1 activity and FAD3 activity, and combinations of these deficiencies on the net accumulation of hydroxyl-fatty acids in transgenic Arabidopsis. 
The other issue that these works address relates to the configuration of the substrate for the fatty acid modifying enzymes. The fatty acid modifying enzymes, whether the hydroxylase or the cyclopropane synthase or other fatty acid modifying enzymes, require specific configurations of their molecular substrates. FIG. 1 of Smith, et al. (2003) notes “For convenience, fatty acids are shown as free fatty acids.” (emphasis added) The fatty acid synthase/fatty acid modifying enzymes act upon their fatty acid substrate when the substrate is configured in an esterified form of one sort or another. The diagram of the options for incorporation of hydroxyl fatty acid (HFA) into HFA-TAG shown in van Erp, et al. (2011), FIG. 1, serves to point out the array of pathways, enzymes and substrate pools that participate in the desired outcome of balancing synthesis, degradation and conversion to HFA-TAG. Thus, as described in van Erp, et al. (2011) and Browse et al. (U.S. Pat. No. 8,101,818), selection of the additional activity (or activities) to be co-expressed in the prospective transgenic, mFA-producing crop plant is not a trivial undertaking. Because of the interacting and intersecting pathways, the effective combination that produces the outcome of significant accumulation of the desired mFA in the seeds (or other tissues) of the targeted crop plant cannot be predicted or foreseen. Simply stating, for example, that “co-expression of a suitable acyltransferase” (lank, et al., U.S. Pat. No. 7,723,574) would make it possible to increase accumulation of a modified fatty acid in transgenic plants does not solve the problem of how to select the suitable acyltransferase from among the numerous potential candidates.
Thus, there remains a need to produce a transgenic crop plant that accumulates commercially relevant amounts of modified fatty acids of interest and particularly in the present invention, cyclopropane fatty acids. In addition to the cyclopropane fatty acid synthase, the metabolic backgrounds of the progenitor plant, and the definition of the acyltransferase or other enzymes to be co-expressed with the cyclopropane fatty acid synthase represent aspects of the present invention.